The specific focus of this proposal is to define the transforming growth factor-n (TGF-B) signaling pathway in gut epithelial cells. The human gut epithelium is maintained by a continuous process of self-renewal, which requires a balance between cell division and apoptosis. Alteration in gut renewal is a hallmark of many diseases of the gut, such as neoplastic growth and inflammatory bowel diseases, all of which contribute to a major health care burden for the United States' population. Progress in the treatment of these diseases has been hampered by the lack of insight into the molecular mechanisms regulating gut renewal. TGF-B is a most important physiological regulator of gut renewal. TGF-B signal is transduced from the receptor complex to the nucleus through a novel class of proteins, the Smads. The goal of this proposal is to determine whether TGF-f3 regulation of cell division and apoptosis is mediated through Smad proteins. We have planned experiments with the following two specific aim. In specific aim 1, we will characterize the role of Smads in TGF-B regulation of cell division. We will determine 1) which endogenous Smads are activated by TGF-B in gut epithelial cells 2) whether endogenous Smads are essential for TGF-B signaling in gut epithelial cells, and 3) whether endogenous Smads are essential for TGF-B inhibition of cyclin Dl expression, Cdk4 activity and cell cycle progression. In specific aim 2, we will characterize the role of Smads in TGF-B-induced apoptosis. We will determine 1) whether Smad3 expression correlates with sensitivity to TGF-B-induced apoptosis, 2) whether Smad3 is essential for TGF-B-induced apoptosis in gut epithelial cells, 3) which domain(s) of Smad3 is required for TGF-B-induced apoptosis, 4) which structural differences between Smad2 and Smad3 are responsible for different apoptosis responses, and 5) the changes in gene expression in TGF-B-induced apoptosis. The studies in the current proposal will extend our previous findings by further delineating the molecular mechanisms that mediate TGF-B's effects in the gut. By delineating the role of Smads in the gut, we hope to determine whether they are potential therapeutic targets for the management of human diseases of the gut.